! Project Summary Millions of Americans today have an opioid use disorder (OUD). Millions more misuse opioids, and the crisis continues to grow. The goal of this proposal is to speed the discovery of non-addictive analgesics by providing drug discovery teams with simpler, more robust, more quantitative, assays for agonist bias. Driven by the urgency of the problem we are seeking Fast Track support to create new assay and analytic tools for drug discovery in OUD research. Our goal is to optimize and test new assays for agonist bias at particular receptors that couple to both the Gi and ?-arrestin signaling pathway, and create new tools to improve the analysis of structure/activity relationships. There are good reasons to search for biased agonists to the receptors identified in the NIDA ?top ten? list of medication development priorities. Biased agonists could activate beneficial signaling pathways while avoiding those that cause adverse effects. Finding these biased agonists is difficult: current assays for detecting bias, while established and validated, suffer from drawbacks that are limiting translatability to animal models and clinical studies. These include entirely different sets of experimental conditions for measuring the different signaling pathways being compared and different time courses of the response being measured. The latter results in time-dependence of the bias measurement which complicates predictions of in vivo efficacy and complicates SAR tables by adding extra variables. Our new assay will simultaneously measure the kinetics of Gi and ?-arrestin signaling in living cells. This project will involved creating new tools as well as re-purposing ones we have already developed to study non-OUD drug targets. The assay will be optimized for use on standard fluorescence plate readers, and a data analysis toolbox will be developed to simplify quantification of agonist bias based on kinetic measurements. Phase I will complete the initial validation studies on the NOP opioid receptor, with goal of demonstrating assay reliability and sensitivity milestones. Phase II will optimize the assay for D3 dopamine, CB1 cannabinoid and OPRM1 opioid receptors and develop the analysis toolbox for deployment on standard plate readers and software packages commonly used in drug discovery. In the second half of Phase II, assays with detailed protocols will be ready distribute to researchers who are developing new drugs for OUD.